Effect of lentivirus-mediated survivin transfection on the morphology and apoptosis of nucleus pulposus cells derived from degenerative human disc in vitro

Ma,Xuexiao; Lin,Yazhou; Yang,Kun; Yue,Bin; Xiang,Hongfei; Chen,Bohua

doi:10.3892/ijmm.2015.2225

Effect of lentivirus-mediated survivin transfection on the morphology and apoptosis of nucleus pulposus cells derived from degenerative human disc in vitro

Authors:
- Xuexiao Ma
- Yazhou Lin
- Kun Yang
- Bin Yue
- Hongfei Xiang
- Bohua Chen
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Affiliations: Department of Orthopedic Surgery, The Affiliated Hospital of Qingdao University Medical College, Qingdao University, Qingdao, Shandong 266003, P.R. China, Medical Research Center, The Affiliated Hospital of Qingdao University Medical College, Qingdao University, Qingdao, Shandong 266003, P.R. China
Published online on: May 26, 2015 https://doi.org/10.3892/ijmm.2015.2225
Pages: 186-194
Copyright: © Ma et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY_NC 3.0].

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Abstract

Lower back pain is a common concern, and 40% of all cases involve the degeneration of the intervertebral disc (IVD). However, the excessive apoptosis of disc cells plays an important role in IVD degeneration, particularly in the nucleus pulposus (NP). Thus, anti-apoptotic gene therapy to attenuate or reverse the degenerative process within the NP is being developed. Survivin is a unique inhibitor of apoptosis (IAP) and has been extensively investigated in cancer cells. However, little is known of the effects of survivin transfection on NP cells derived from degenerative human disc. In this study, we aimed to investigate the effects of lentivirus (LV)‑mediated survivin transfection on the morphology and apoptosis of NP cells derived from degenerative human disc in vitro. NP cells were transfected with LV‑mediated survivin. Subsequently, cell morphology was observed and the survivin mRNA expression levels were measured by RT‑qPCR. Apoptosis was analyzed by flow cytometry and by measuring caspase‑3 activity. The results revealed that the morphology of the NP cells derived from degenerative human disc transfected with LV‑mediated survivin was significantly altered as evidenced by cytomorphosis, the reduction of the cytoplasm and cell shrinkage. Following transfection, survivin gene expression significantly increased in the transfected cells and subsequent generation cells; however, no significant differences in the cell apoptotic rate and caspase‑3 activity were observed. We found that transfection of the survivin gene into NP cells led to the stable expression of survivin and induced marked changes in cell morphology. Furthermore, no significant anti-apoptotic effects were observed following LV‑mediated survivin transfection. Overall, our findings demonstrate that LV carrying surviving may be used to successfully enforce the expression of survivin in NP cells. However, cell morphology was evidently altered, whereas the apoptotic rate did not decrease. Comprehensive studies on the feasibility of using survivin in gene therapy in an aim to attenuate disc degeneration are warranted. Further research on the mechanisms responsible for the changes in cell morphology and cell function are also required.

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1	McMeeken J, Tully E, Stillman B, Nattrass C, Bygott IL and Story I: The experience of back pain in young Australians. Man Ther. 6:213–220. 2001. View Article : Google Scholar : PubMed/NCBI
2	Waddell G: Low back disability. A syndrome of Western civilization. Neurosurg Clin N Am. 2:719–738. 1991.PubMed/NCBI
3	Waddell G: Low back pain: A twentieth century health care enigma. Spine (Phila Pa 1976). 21:2820–2825. 1996. View Article : Google Scholar
4	Hillman M, Wright A, Rajaratnam G, Tennant A and Chamberlain MA: Prevalence of low back pain in the community: Implications for service provision in Bradford, UK. J Epidemiol Community Health. 50:347–352. 1996. View Article : Google Scholar : PubMed/NCBI
5	Hoy D, Brooks P, Blyth F and Buchbinder R: The Epidemiology of low back pain. Best Pract Res Clin Rheumatol. 24:769–781. 2010. View Article : Google Scholar
6	Juniper M, Le TK and Mladsi D: The epidemiology, economic burden, and pharmacological treatment of chronic low back pain in France, Germany, Italy, Spain and the UK: A literature-based review. Expert Opin Pharmacother. 10:2581–2592. 2009. View Article : Google Scholar : PubMed/NCBI
7	Rossignol M, Rozenberg S and Leclerc A: Epidemiology of low back pain: What’s new? Joint Bone Spine. 76:608–613. 2009. View Article : Google Scholar : PubMed/NCBI
8	Luoma K, Riihimäki H, Luukkonen R, Raininko R, Viikari-Juntura E and Lamminen A: Low back pain in relation to lumbar disc degeneration. Spine (Phila Pa 1976). 25:487–492. 2000. View Article : Google Scholar
9	Levicoff EA, Gilbertson LG and Kang JD: Gene therapy for disc repair. Spine J. 5(Suppl 6): 287S–296S. 2005. View Article : Google Scholar : PubMed/NCBI
10	Roughley PJ: Biology of intervertebral disc aging and degeneration: Involvement of the extracellular matrix. Spine (Phila Pa 1976). 29:2691–2699. 2004. View Article : Google Scholar
11	Singh K, Masuda K, Thonar EJ, An HS and Cs-Szabo G: Age-related changes in the extracellular matrix of nucleus pulposus and anulus fibrosus of human intervertebral disc. Spine (Phila Pa 1976). 34:10–16. 2009. View Article : Google Scholar
12	Sivan SS, Hayes AJ, Wachtel E, Caterson B, Merkher Y, Maroudas A, Brown S and Roberts S: Biochemical composition and turnover of the extracellular matrix of the normal and degenerate intervertebral disc. Eur Spine J. 23(Suppl 3): S344–S353. 2014. View Article : Google Scholar
13	Freemont AJ: The cellular pathobiology of the degenerate intervertebral disc and discogenic back pain. Rheumatology (Oxford). 48:5–10. 2009. View Article : Google Scholar
14	Le Maitre CL, Freemont AJ and Hoyland JA: The role of interleukin-1 in the pathogenesis of human intervertebral disc degeneration. Arthritis Res Ther. 7:R732–R745. 2005. View Article : Google Scholar : PubMed/NCBI
15	Smith LJ, Nerurkar NL, Choi KS, Harfe BD and Elliott DM: Degeneration and regeneration of the intervertebral disc: Lessons from development. Dis Model Mech. 4:31–41. 2011. View Article : Google Scholar :
16	Rannou F, Lee TS, Zhou RH, Chin J, Lotz JC, Mayoux-Benhamou MA, Barbet JP, Chevrot A and Shyy JY: Intervertebral disc degeneration: The role of the mitochondrial pathway in annulus fibrosus cell apoptosis induced by overload. Am J Pathol. 164:915–924. 2004. View Article : Google Scholar : PubMed/NCBI
17	Zhao CQ, Liu D, Li H, Jiang LS and Dai LY: Interleukin-1beta enhances the effect of serum deprivation on rat annular cell apoptosis. Apoptosis. 12:2155–2161. 2007. View Article : Google Scholar : PubMed/NCBI
18	Scaffidi C, Schmitz I, Zha J, Korsmeyer SJ, Krammer PH and Peter ME: Differential modulation of apoptosis sensitivity in CD95 type I and type II cells. J Biol Chem. 274:22532–22538. 1999. View Article : Google Scholar : PubMed/NCBI
19	Ha KY, Kim BG, Kim KW, Oh IS and Seo JY: Apoptosis in the sequestrated nucleus pulposus compared to the remaining nucleus pulposus in the same patient. Spine (Phila Pa 1976). 36:683–689. 2011. View Article : Google Scholar
20	Kaneyama S, Nishida K, Takada T, Suzuki T, Shimomura T, Maeno K, Kurosaka M and Doita M: Fas ligand expression on human nucleus pulposus cells decreases with disc degeneration processes. J Orthop Sci. 13:130–135. 2008. View Article : Google Scholar : PubMed/NCBI
21	Kuo YJ, Wu LC, Sun JS, Chen MH, Sun MG and Tsuang YH: Mechanical stress-induced apoptosis of nucleus pulposus cells: An in vitro and in vivo rat model. J Orthop Sci. 19:313–322. 2014. View Article : Google Scholar
22	Park JB, Lee JK, Park EY and Riew KD: Fas/FasL interaction of nucleus pulposus and cancer cells with the activation of caspases. Int Orthop. 32:835–840. 2008. View Article : Google Scholar
23	Johnson ME and Howerth EW: Survivin: A bifunctional inhibitor of apoptosis protein. Vet Pathol. 41:599–607. 2004. View Article : Google Scholar : PubMed/NCBI
24	Sah NK, Khan Z, Khan GJ and Bisen PS: Structural, functional and therapeutic biology of survivin. Cancer Lett. 244:164–171. 2006. View Article : Google Scholar : PubMed/NCBI
25	Andersen MH, Svane IM, Becker JC and Straten PT: The universal character of the tumor-associated antigen survivin. Clin Cancer Res. 13:5991–5994. 2007. View Article : Google Scholar : PubMed/NCBI
26	Ghadimi MP, Young ED, Belousov R, et al: Survivin is a viable target for the treatment of malignant peripheral nerve sheath tumors. Clin Cancer Res. 18:2545–2557. 2012. View Article : Google Scholar : PubMed/NCBI
27	Romagnoli M, Séveno C, Bataille R and Barillé-Nion S: Survivin in cancerology : Molecular aspects and therapeutic applications. Med Sci (Paris). 24:821–827. 2008.In French. View Article : Google Scholar
28	Lechler P, Balakrishnan S, Schaumburger J, Grässel S, Baier C, Grifka J, Straub RH and Renkawitz T: The oncofetal gene survivin is re-expressed in osteoarthritis and is required for chondrocyte proliferation in vitro. BMC Musculoskelet Disord. 12:1502011. View Article : Google Scholar : PubMed/NCBI
29	Baran M, Möllers LN, Andersson S, Jonsson IM, Ekwall AK, Bjersing J, Tarkowski A and Bokarewa M: Survivin is an essential mediator of arthritis interacting with urokinase signalling. J Cell Mol Med. 13:3797–3808. 2009. View Article : Google Scholar : PubMed/NCBI
30	Bokarewa M, Tarkowski A and Magnusson M: Pathological survivin expression links viral infections with pathogenesis of erosive rheumatoid arthritis. Scand J Immunol. 66:192–198. 2007. View Article : Google Scholar : PubMed/NCBI
31	Yang KS, Yue B, Ma XX, et al: The expression of survivin and its significance in fetal intervertebral disc. Qingdao Daxue Yixueyuan Xuebao. 49:205–206. 2013.In Chinese.
32	Yang KS: The expression of survivin and its significance in intervertebral disc. PhD thesis. Qingdao University; 2013
33	Zucchini-Pascal N, de Sousa G and Rahmani R: Lindane and cell death: At the crossroads between apoptosis, necrosis and autophagy. Toxicology. 256:32–41. 2009. View Article : Google Scholar
34	Kluba T, Niemeyer T, Gaissmaier C and Gründer T: Human anulus fibrosis and nucleus pulposus cells of the intervertebral disc: Effect of degeneration and culture system on cell phenotype. Spine (Phila Pa 1976). 30:2743–2748. 2005. View Article : Google Scholar
35	Le Maitre CL, Pockert A, Buttle DJ, Freemont AJ and Hoyland JA: Matrix synthesis and degradation in human intervertebral disc degeneration. Biochem Soc Trans. 35:652–655. 2007. View Article : Google Scholar : PubMed/NCBI
36	Nerlich AG, Schleicher ED and Boos N: 1997 Volvo Award winner in basic science studies. Immunohistologic markers for age-related changes of human lumbar intervertebral discs. Spine (Phila Pa 1976). 22:2781–2795. 1997. View Article : Google Scholar
37	Sive JI, Baird P, Jeziorsk M, Watkins A, Hoyland JA and Freemont AJ: Expression of chondrocyte markers by cells of normal and degenerate intervertebral discs. Mol Pathol. 55:91–97. 2002. View Article : Google Scholar : PubMed/NCBI
38	Cui S, Wienhoefer N and Bilitewski U: Genistein induces morphology change and G2/M cell cycle arrest by inducing p38 MAPK activation in macrophages. Int Immunopharmacol. 18:142–150. 2014. View Article : Google Scholar
39	Astuti P, Pike T, Widberg C, Payne E, Harding A, Hancock J and Gabrielli B: MAPK pathway activation delays G2/M progression by destabilizing Cdc25B. J Biol Chem. 284:33781–33788. 2009. View Article : Google Scholar : PubMed/NCBI
40	Gil-Araujo B, Toledo Lobo MV, Gutiérrez-Salmerón M, Gutiérrez-Pitalúa J, Ropero S, Angulo JC, Chiloeches A and Lasa M: Dual specificity phosphatase 1 expression inversely correlates with NF-κB activity and expression in prostate cancer and promotes apoptosis through a p38 MAPK dependent mechanism. Mol Oncol. 8:27–38. 2014. View Article : Google Scholar
41	Pereira L, Igea A, Canovas B, Dolado I and Nebreda AR: Inhibition of p38 MAPK sensitizes tumour cells to cisplatin-induced apoptosis mediated by reactive oxygen species and JNK. EMBO Mol Med. 5:1759–1774. 2013. View Article : Google Scholar : PubMed/NCBI
42	Wang H, Ye Y, Chui JH, Zhu GY, Li YW, Fong DW and Yu ZL: Oridonin induces G2/M cell cycle arrest and apoptosis through MAPK and p53 signaling pathways in HepG2 cells. Oncol Rep. 24:647–651. 2010.PubMed/NCBI
43	Carter BZ, Mak DH, Schober WD, Cabreira-Hansen M, Beran M, McQueen T, Chen W and Andreeff M: Regulation of survivin expression through Bcr-Abl/MAPK cascade: Targeting survivin overcomes imatinib resistance and increases imatinib sensitivity in imatinib-responsive CML cells. Blood. 107:1555–1563. 2006. View Article : Google Scholar
44	Anderson DG and Tannoury C: Molecular pathogenic factors in symptomatic disc degeneration. Spine J. 5(Suppl 6): 260S–266S. 2005. View Article : Google Scholar : PubMed/NCBI
45	Kim KW, Lim TH, Kim JG, Jeong ST, Masuda K and An HS: The origin of chondrocytes in the nucleus pulposus and histologic findings associated with the transition of a notochordal nucleus pulposus to a fibrocartilaginous nucleus pulposus in intact rabbit intervertebral discs. Spine (Phila Pa 1976). 28:982–990. 2003. View Article : Google Scholar
46	Buckwalter JA: Aging and degeneration of the human intervertebral disc. Spine (Phila Pa 1976). 20:1307–1314. 1995.